In vitro high frequency multiplication and assessment of genetic fidelity of Corallocarpus epigaeus: an endangered medicinal plant

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Research Articles | Published:

Print ISSN : 0970-4078.
Online ISSN : 2229-4473.
Pub Email:
Doi: 10.1007/s42535-019-00085-6
First Page: 63
Last Page: 73
Views: 1995

Keywords: Corallocarpus epigaeus , Nodal explants, Micropropagation, ISSR, Genetic fidelity


Corallocarpus epigaeus (Rottler) Hook.f. is an endangered tuberous medicinal climber of family Cucurbitaceae. Despite high medicinal value, over-exploitation made it threatened. In vitro propagation has been adopted for conserving this endangered medicinal plant. Direct shoots induction was achieved from nodal explants on MS medium fortified with various concentrations of BAP and TDZ individually and BAP + IAA, TDZ + IAA, BAP + l-glutamic acid and TDZ + l-glutamic acid combinations. The highest frequency of multiple shoots (43.33 ± 0.53) was achieved on MS medium fortified with 1.5 mg/l TDZ + 1.5 mg/l IAA from nodal explants but shoot length (12.9 ± 0.15 cm) was high on MS medium supplemented with 1.0 mg/l TDZ and 2.0 mg/l l-Glutamic acid. The highest percentage (78%) of rooting was achieved on half strength MS medium augmented with 1.0 mg/l IBA with a mean number of roots 10.76 ± 0.30 cm, an average root length is 1.69 ± 0.07 cm. Rooted plantlets were acclimatized in the greenhouse and successfully transplanted to natural conditions with a 68% survival rate. ISSR markers were used to check the genetic fidelity between in vivo and in vitro developed plantlets. The results indicated that the micropropagated plants are monomorphic and true type when compare with mother plant.

                Corallocarpus epigaeus
              , Nodal explants, Micropropagation, ISSR, Genetic fidelity

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  1. Ahuja MR (1993) Micropropagation à la carte. In: Micropropagation of woody plants. Springer, Dordrecht, pp 3–9

  2. Ambethkar A, Umamaheswari C, Margaret S, Sivanandhan G, Selvaraj N (2012) In vitro regeneration of multiple shoots from cotyledon explants of Trichosanthes anguina L. (Snake gourd). Indian J Nat Sci 2(10):833–840

  3. Anand SP, Jeyachandran R (2004) In vitro multiple shoot regeneration from nodal explants of Zehneria scabra (L.f) Sonder—an important medicinal climber. Plant Tissue Cult 14(2):101–106

  4. Anil MNV, Kumari K, Wate SR (2014) Loss of biodiversity and conservation strategies: an outlook of Indian scenario. Asian J Conserv Biol 3:105–114

  5. Arulanandam L, Kumar SG, Sowmini M (2011a) Micropropagation and conservation of rare medicinal plant Wattakaka volubilis (Linn.) Stapf. Indian J Biotechnol 10:238–241

  6. Arulanandam L, Peter J, Ghanthikumar S (2011b) Indirect organogenesis of Vitex trifolia Linn.—an important medicinal plant. Indian J Nat Prod Res 2(2):261–264

  7. Arulanandam L, Peter J, Ghanthikumar S (2011c) Indirect organogenesis of Vitex trifolia Linn.—an important medicinal plant. Indian J Nat Prod Res 2(2):261–264

  8. Atal CK, Kapur BM (1982) Cultivation and utilization of aromatic plants. Regional research laboratory, CSIR, Jammu-Tawi

  9. Benelli C, Fabbri A, Grassi S, Lambardi M, Rugini E (2001) Histology of somatic embryogenesis in mature tissues of olive (Olea europaea L.). J Hortic Sci Biotechnol 76(1):112–119

  10. Bhardwaj R, Dutta S, Sharma KC (2011) Conserving biodiversity of medicinal plants from central Aravallis of Rajasthan, India. J Environ Res Dev 6(1):69–75

  11. Bohidar S, Thirunavoukkarasu M, Rao TV (2008) Effect of plant growth regulators on in vitro micropropagation of “Garden Rue”(Ruta graveolens L.). Int J Integr Biol 3(1):36–43

  12. Choudhary K, Singh M, Pillai U (2008) Ethnobotanical survey of Rajasthan—an update. Am Euras J Bot 1(2):38–45

  13. Das T, Mitra GC (1990) Micropropagation of Eucalyptus tereticornis Smith. Plant Cell Tissue Organ Cult 22(2):95–103

  14. Devendra NK, Rajanna L, Sheetal C, Seetharam YN (2008) In vitro clonal propagtion of Trichosanthes cucumerina L. var. cucumerina. Plant Tissue Cult Biotechnol 18(2):103–111

  15. Dhabhai K, Sharma MM, Batra A (2010) In vitro clonal propagation of Acacia nilotica (L.)—a nitrogen fixing tree. Researcher 2(3):7–11

  16. Dhir R, Shekhawat GS (2012) Critical review on Tecomella undulata: a medicinally potent endangered plant species of Indian Thar Desert. Int J Curr Res 4(6):36–44

  17. Dhir R, Shekhawat GS (2013) Production, storability and morphogenic response of alginate encapsulated axillary meristems and genetic fidelity evaluation of in vitro regenerated Ceropegia bulbosa: a pharmaceutically important threatened plant species. Ind Crops Prod 47:139–144

  18. Dhir R, Shekhawat GS, Alam A (2014) Improved protocol for somatic embryogenesis and calcium alginate encapsulation in Anethum graveolens L.: a medicinal herb. Appl Biochem Biotechnol 173(8):2267–2278

  19. Doyle JJ, Doyle JL (1990) Isolation ofplant DNA from fresh tissue. Focus 12(13):39–40

  20. Gomez KA, Gomez AA (1976) Stastical procedure from agricultural research emphasis on Rice. International rice research inistute, Los Banos, Philippines

  21. Govind KR, Major S, Neha PR, Bhardwaj DR, Sanjeev K (2012) In vitro propagation of spine gourd (Momordica dioica Roxb.) and assessment of genetic fidelity of micropropagated plants using RAPD analysis. Physiol Mol Biol Plants 18(3):273–280

  22. Hoque A, Islam R, Joarder OI (1995) In vitro plantlets differentiation in kakrol (Momordica dioica Roxb). Plant Tissue Cult 5(2):119–124

  23. Jain R, Sinha A, Jain D, Kachhwaha S, Kothari SL (2011) Adventitious shoot regeneration and in vitro biosynthesis of steroidal lactones in Withania coagulans (Stocks) Dunal. Plant Cell Tissue Organ Cult 105(1):135–140

  24. Jana S, Shekhawat GS (2011) Critical review on medicinally potent plant species: Gloriosa superba. Fitoterapia 82(3):293–301

  25. Jeong MJ, Song HJ, Park DJ, Min JY, Jo JS, Kim BM, Kim HG, Kim YD, Kim RM, Karigar CS, Choi MS (2009) High frequency plant regeneration from abnormal shoot organogenesis in medicinal tree Hovenia dulcis. Plant Cell Tiss Organ Cult 98:59–65

  26. John Peter Arulanandam L, Ghanthikumar S, Sowmini M (2011) Micropropagation and conservation of rare medicinal plant Watakaka volubilis (Linn.). Stapf Indian J Biotech 10:238–241

  27. Joshi K, Chavan P, Warude D, Patwardhan B (2004) Molecular markers in herbal drug technology. Curr Sci 87:159–165

  28. Khatun MM, Hossain MS, Haque MA, Khalekuzzaman M (2010) In vitro propagation of Citrullus lanatus Thumb. from nodal explants culture. J Bangladesh Agric Univ 8(2):203–206

  29. Kour B, Kour G, Kaul S, Dhar MK (2015) In vitro mass multiplication and assessment of genetic stability of in vitro raised Artemisia absinthium L. plants using ISSR and SSAP molecular markers. Adv Bot 2014

  30. Krug MGZ, Stipp LCL, Rodriguez APM, Mendes BMJ (2005) In vitro organogenesis in watermelon cotyledons. Pesq Agropec Bras 40:861–865

  31. Kumar S, Singh N (2009) Micropropagation of Prosopis cineraria (l.) Druce—a multipurpose desert tree. Researcher 1(3):9–13

  32. Kumar HA, Murthy HN, Paek KY (2003) Embryogenesis and plant regeneration from anther cultures of Cucumis sativus L. Sci Hortic 98(3):213–222

  33. Kundu S, Salma U, Ali MN, Mandal N (2017) Factors influencing large-scale micropropagation of Sphagneticola calendulacea (L.) Pruski and clonality assessment using RAPD and ISSR markers. In Vitro Cell Dev Biol Plant 53(3):167–177

  34. Larkin PJ, Scowcroft WR (1981) Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60(4):197–214

  35. Mathur S, Shekhawat GS, Batra A (2002) An efficient in vitro method for mass propagation of Salvadora persica via apical meristem. J Plant Biochem Biotechnol 11(2):125–127

  36. Moreno V, Garcia-Sogo M, Granell I, Garcia-Sogo B, Roig LA (1985) Plant regeneration from calli of melon (Cucumis melo L., cv.‘Amarillo Oro’). Plant Cell Tissue Organ Cult 5(2):139–146

  37. Murthy KSR, Ravindranath D, Sandhya RS, Pullaiah T (2013) Ethnobotany and distribution of wild and cultivated genetic resources of Cucurbitaceae in the Eastern Ghats of Peninsular India. Top J Herb Med 2(6):149–158

  38. Mustafa M, Swamy TN, Raju S, Mohammad SP (2013) Multiple shoot induction from the nodal cultures of teasle gourd (Momordica dioica Roxb.). Int J Biosci 3:8–12

  39. Nabi SA, Rashid MM, Al-Amin M, Rasul MG (2002) Organogenesis in teasel gourd (Momordica dioica Roxb.). Plant Tissue Cult 12:173–180

  40. Nadkarni KM (1982) The Indian materia medica, vol I. Popular Prakashan, Bombay, p 377

  41. Nanda RM, Das P, Rout GR (2004) In vitro clonal propagation of Acacia mangium Willd and its evaluation of genetic stability through RAPD marker. Ann For Sci 61(4):381–386

  42. Narayan JP (2016) Ex-situ conservation of the rare and threatened medicinal climber Corallocarpus epigaeus Rottler through in vitro regeneration method. Biotechnol J Int 1:1–10

  43. Nayak SA, Kumar S, Satapathy K, Moharana A, Behera B, Barik DP, Naik SK (2013) In vitro plant regeneration from cotyledonary nodes of Withania somnifera (L.) Dunal and assessment of clonal fidelity using RAPD and ISSR markers. Acta Physiol Plant 35(1):195–203

  44. Niranjan MH, Sudarshana MS, Girisha ST (2010) In vitro multiple shoot induction from excised shoot tips and nodal segment explants of-Lagerstroemia indica (L)—a medicinal cum ornamental shrub. J Biomed Sci Res 2(3, Cop):212–217

  45. Oldfield S (1997) Cactus and succulent plants: status survey and conservation action plan. International Union for Conservation of Nature and Natural Resources (IUCN)

  46. Pal SP, Alam I, Anisuzzaman M, Sarker KK, Sharmin SA, Alam MF (2007) Indirect organogenesis in summer squash (Cucurbita pepo L.). Turkish J Agric For 31(1):63–70

  47. Palni LMS (ed) (2012) Compendium on Indian biosphere reserves: progression during two decades of conservation. GB Pant Institute of Himalayan Environment & Development

  48. Pandey RN, Singh SP, Rastogi J, Sharma ML, Singh RK (2012) Early assessment of genetic fidelity in sugarcane (Saccharum officinarum) plantlets regenerated through direct organogenesis with RAPD and SSR markers. Aust J Crop Sci 6:618–624

  49. Raghu E, Muralikrishna N, Srinivas K, Bharathkumar K, Yashodhara V, Pandarinath S, Venkateswar Rao A (2016) An efficient and high frequency regeneration protocol in two cultivars of Capsicum annuum L. cvs. G3 and G4. Int J Curr Biotechnol 4(3):1–8

  50. Ramakrishnan M, Ceasar SA, Duraipandiyan V, Ignacimuthu S (2014) Efficient plant regeneration from shoot apex explants of maize (Zea mays) and analysis of genetic fidelity of regenerated plants by ISSR markers. Plant Cell Tiss Organ Cult 119:183–196

  51. Rani V, Parida A, Raina S (1995) Random amplified polymorphicDNA (RAPD) markers for genetic analysis in micropropagated plants of Populus deltoids Marsh. Plant Cell Rep 14:459–462

  52. Rathore MS, Mastan SG, Yadav P, Bhatt VD, Shekhawat NS, Chikara J (2015) Shoot regeneration from leaf explants of Withania coagulans (Stocks) Dunal and genetic stability evaluation of regenerates with RAPD and ISSR markers. S Afr J Bot 102:12–17

  53. Rohela GK, Jogam P, Shabnam AA, Shukla P, Abbagani S, Ghosh MK (2018) In vitro regeneration and assessment of genetic fidelity of acclimated plantlets by using ISSR markers in PPR-1 (Morus sp.): an economically important plant. Sci Hortic 241:313–321

  54. Rohela GK, Jogam P, Bylla P, Reuben C (2019) Indirect regeneration and assessment of genetic fidelity of acclimated plantlets by SCoT, ISSR, and RAPD markers in Rauwolfia tetraphylla L.: an endangered medicinal plant. BioMed Res Int

  55. Rout GR, Senapati SK, Aparajita S, Palai SK (2009) Studies on genetic identification and genetic fidelity of cultivated banana using ISSR markers. Plant Omics 2(6):250

  56. Saeed T, Shahzad A, Ahmad N, Parveen S (2018) High frequency conversion of non-embryogenic synseeds and assessment of genetic stability through ISSR markers in Gymnema sylvestre. PCTOC 134(1):163–168

  57. Saha S, Sengupta C, Roy S, Ghosh P (2014) Micropropagation and analysis of genetic stability in regenerated plantlets of Ocimum canum Sims. Ind J Plant Physiol 19(2):174–183

  58. Sarowar S, Oh HY, Hyung NI, Min BW, Harn CH, Yang SK, Ok SH, Shin JS (2003) In vitro micropropagation of a Cucurbita interspecific hybrid cultivar—a root stock plant. Plant Cell Tiss Org Cult 75:179–182

  59. Sharma SK (2009) Medicinal plants: a probe in the forests of Rajasthan. In: Trivedi PC (ed) Medicinal plants: utilization and conservation, 2nd edn. Avishkar Publishers, Jaipur, pp 181–216

  60. Sharma MM, Batra A (2006) High frequency plantlet regeneration in Indian Ginseng: Withania somnifera L. (Dunal). Physiol Mol Biol Plants 12(4):289

  61. Shekhawat GS, Mathur S, Batra A (2009) Role of phytohormones and nitrogen in somatic embryogenesis induction in cell culture derived from leaflets of Azadirachta indica. Biol Plant 53(4):707

  62. Sivaram L, Mukundan U (2003) In vitro culture studies on Stevia rebaudiana. In Vitro Cell Dev Biol Plant 39(5):520–523

  63. Sivkumar T, Kannan K, Manavalan R (2009) Pharmacognostical investigations of Corallocarpus epigaeus (Rottler) CB Clark. Lateral 2(1):159–166

  64. Slazak B, Sliwinska E, Saługa M, Ronikier M, Bujak J, Słomka A, Goransson U, Kuta E (2015) Micropropagation of Viola uliginosa (Violaceae) for endangered species conservation and for somaclonal variation-enhanced cyclotide biosynthesis. Plant Cell Tiss Org Cult 120(1):179–190

  65. Stipp LCL, Mendes BMJ, Piedade SMS, Rodriguez APM (2001) In vitro morphogenesis of Cucumis melo var. inodorus. Plant Cell Tissue Organ Cult 65(1):81–89

  66. Sultana RS, Bari Miah MA (2003) In vitro propagation of karalla (Momordica charantia Linn.) from nodal segment and shoot tip. J Biol Sci 3(12):1134–1139

  67. Sultana RS, Bari MA, Rahman MH, Rahman MM, Siddique NA, Khatun N (2004) In vitro rapid regeneration of plantlets from leaf explant of water melon (Citrullus lanatus Thumb.). Biotechnology 3(2):131–135

  68. Tanimoto E (2005) Regulation of root growth by plant hormones-roles for auxin and gibberellin. Crit Rev Plant Sci 24(4):249–265

  69. Thiruvengadam M, Rekha KT, Yang CH, Jayabalan N, Chung IM (2010) High-frequency shoot regeneration from leaf explants through organogenesis in bitter melon (Momordica charantia L.). Plant Biotechnol Rep 4(4):321–328

  70. Thomas TD, Sreejesh KR (2004) Callus induction and plant regeneration from cotyledonary explants of ash gourd (Benincasa hispida L.). Sci Hortic 100:359–367

  71. Tiwari JK, Chandel P, Gupta S, Gopal J, Singh BP (2013) Analysis of genetic stability of in vitro propagated potato microtubers using DNA markers. Physiol Mol Biol Plants 19(4):587–595

  72. Wagh VV, Jain AK (2013) Status of threatened medicinal plants of Jhabua district, Madhya Pradesh, India. Ann Plant Sci 2(10):395–400

  73. Xing Y, Yu Y, Luo X, Zhang JN, Zhao B, Guo YD (2010) High efficiency organogenesis and analysis of genetic stability of the regenerants in Solanum melongena. Biol Plant 54(2):231–236

  74. Yang XM, An LZ, Xiong YC, Zhang JP, Li Y, Xu SJ (2008) Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine. Biol Plant 52(2):209–214



The authors are grateful to Prof. Sadanandam Abbagani Formar Dean Faculty of Sciences and Dr. A. V. Rao Department of Biotechnology, Kakatiya University for their valuable suggestions and encouragement. The authors are thankful to Dr. T. Christopher Reuben Head, Department of Botany, Dr. T. Shasthree Head, Department of Biotechnology, Kakatiya University, warangal for facilities and Mr. Ramesh Kandagatla for his help with setting the plant images.

Author Information

Vemula Suresh
Plant Tissue Culture and Molecular Taxonomy Laboratory, Department of Botany, Kakatiya University, Warangal, India

Koppula Thirupathi
Plant Tissue Culture and Molecular Taxonomy Laboratory, Department of Botany, Kakatiya University, Warangal, India

Jogam Phanikanth
Department of Biotechnology, Kakatiya University, Warangal, India

Mohammed Mustafa
Plant Tissue Culture and Molecular Taxonomy Laboratory, Department of Botany, Kakatiya University, Warangal, India